Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis.

With the exception of the methanogenic archaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum deltaH, all organisms surveyed contain orthologs of Escherichia coli cysteinyl-tRNA synthetase (CysRS). The characterization of CysRS-encoding (cysS) genes and the demonstration of their ability to complement an E. coli cysSts mutant reveal that Methanococcus maripaludis and Methanosarcina barkeri, two other methanogenic archaea, possess canonical CysRS proteins. A molecular phylogeny inferred from 40 CysRS sequences indicates that the CysRS of M. maripaludis and Methanosarcina spp. are specific relatives of the CysRS of Pyrococcus spp. and Chlamydia, respectively. This result suggests that the CysRS gene was acquired by lateral gene transfer in at least one euryarchaeotic lineage.

Glutamyl-tRNA(Gln) amidotransferase in Deinococcus radiodurans may be confined to asparagine biosynthesis.

Asparaginyl-tRNA (Asn-tRNA) and glutaminyl-tRNA (Gln-tRNA) are essential components of protein synthesis. They can be formed by direct acylation by asparaginyl-tRNA synthetase (AsnRS) or glutaminyl-tRNA synthetase (GlnRS). The alternative route involves transamidation of incorrectly charged tRNA. Examination of the preliminary genomic sequence of the radiation-resistant bacterium Deinococcus radiodurans suggests the presence of both direct and indirect routes of Asn-tRNA and Gln-tRNA formation. Biochemical experiments demonstrate the presence of AsnRS and GlnRS, as well as glutamyl-tRNA synthetase (GluRS), a discriminating and a nondiscriminating aspartyl-tRNA synthetase (AspRS). Moreover, both Gln-tRNA and Asn-tRNA transamidation activities are present. Surprisingly, they are catalyzed by a single enzyme encoded by three ORFs orthologous to Bacillus subtilis gatCAB. However, the transamidation route to Gln-tRNA formation is idled by the inability of the discriminating D. radiodurans GluRS to produce the required mischarged Glu-tRNAGln substrate. The presence of apparently redundant complete routes to Asn-tRNA formation, combined with the absence from the D. radiodurans genome of genes encoding tRNA-independent asparagine synthetase and the lack of this enzyme in D. radiodurans extracts, suggests that the gatCAB genes may be responsible for biosynthesis of asparagine in this asparagine prototroph.

A euryarchaeal lysyl-tRNA synthetase: resemblance to class I synthetases.

The sequencing of euryarchaeal genomes has suggested that the essential protein lysyl-transfer RNA (tRNA) synthetase (LysRS) is absent from such organisms. However, a single 62-kilodalton protein with canonical LysRS activity was purified from Methanococcus maripaludis, and the gene that encodes this protein was cloned. The predicted amino acid sequence of M. maripaludis LysRS is similar to open reading frames of unassigned function in both Methanobacterium thermoautotrophicum and Methanococcus jannaschii but is unrelated to canonical LysRS proteins reported in eubacteria, eukaryotes, and the crenarchaeote Sulfolobus solfataricus. The presence of amino acid motifs characteristic of the Rossmann dinucleotide-binding domain identifies M. maripaludis LysRS as a class I aminoacyl-tRNA synthetase, in contrast to the known examples of this enzyme, which are class II synthetases. These data question the concept that the classification of aminoacyl-tRNA synthetases does not vary throughout living systems.

Glu-tRNAGln amidotransferase: a novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation.

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

Aminoacyl-tRNA synthesis: divergent routes to a common goal.

Aminoacyl-tRNAs are key components in protein synthesis. They are formed directly by correct acylation of tRNA (by aminoacyl-tRNA synthetases) or indirectly by tRNA-dependent transformation of misacylated tRNAs. The accuracy of aminoacyl-tRNA synthesis is enhanced by a number of further protein-RNA or protein-protein interactions, some of which are restricted to Archaea, and might reflect adaptation mechanisms to diverse conditions.

tRNA-dependent amino acid transformations.

Aminoacyl-tRNAs are either synthesized directly by aminoacyl-tRNA synthetases or indirectly by tRNA-dependent transformation of mischarged tRNAs. The enzymes which participate in the indirect routes may be interesting targets in the development of novel therapeutic compounds. We have purified one such enzyme, Glu-tRNA(Gln) amidotransferase from Bacillus subtilis, to homogeneity and present the initial biochemical characterization data.

tRNA-guanine transglycosylase from Escherichia coli. Minimal tRNA structure and sequence requirements for recognition.

Previously, we have demonstrated that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is capable of utilizing an in vitro generated minihelix consisting of the anticodon stem and loop sequence of E. coli tRNA(Tyr) (Curnow, A. W., Kung, F. L., Koch, K. A., and Garcia, G. A. (1993) Biochemistry 32, 5239-5246). This suggests that the tRNA structural motifs necessary for recognition comprise a loop at the end of a short helix. To gain further insight into the structural requirements for TGT recognition, we have investigated the conformation of this minimal substrate. Thermal denaturation studies and kinetic analyses at 20 and 37 degrees C indicate that this minihelix is predominantly melted at 37 degrees C and that the melted conformation is not a substrate for TGT. This is confirmed by the determination that a non-helical analogue of the minihelix is not a substrate for TGT at either temperature. Two additional minihelices designed to be stable at 37 degrees C, ECYMH (a 4-base pair extension of the previous minihelix) and SCDMH (a yeast tRNA(Asp) analogue of ECYMH), were generated and characterized. Finally, several sequence mutants of SCDMH, focusing on the G30U40 base pair and U33G34U35 loop sequence, have been produced, and kinetic parameter determinations have been performed at 37 degrees C. Our results are consistent with a recent report (Nakanishi, S., Ueda, T., Hori, H., Yamazaki, N., Okada, N., and Watanabe, K. (1994) J. Biol. Chem. 269, 32221-32225) indicating that a UGU sequence in a 7-base loop is the minimal requirement for TGT recognition.

tRNA-guanine transglycosylase from Escherichia coli is a zinc metalloprotein. Site-directed mutagenesis studies to identify the zinc ligands.

tRNA-guanine transglycosylase (TGT) from Escherichia coli catalyzes the exchange of the queuine precursor, preQ1, into tRNA as part of the biosynthetic pathway for the posttranscriptionally modified base, queuine. No significant sequence homologies exist between TGT and any of the proteins in the GenBank database. However, an unusual arrangement of cysteine residues was observed upon manual examination of the TGT sequence. Comparison of this sequence (residues 302-321) revealed similarities to structural zinc-binding motifs in proteins of known structure [Jaffe (1993) Comments Inorg. Chem. 15, 67-93]. Within this region of the TGT sequence, there are six residues (four cysteines and two histidines), any four of which could serve as the ligands to the zinc. We report here that wild-type TGT contains ca. 0.8 mol of zinc/mol of subunit, determined by atomic emission spectrometry. In order to determine which enzyme residues are serving as the ligands to the zinc, site-directed mutagenesis studies have been performed. Gross structural probes (native PAGE and CD spectra), enzyme activity assays, and tRNA-binding assays indicate that cysteines 302, 304, and 307 and histidine 317 are the ligands to the zinc. These results also suggest that the zinc site is necessary for TGT homotrimer formation and for tRNA binding.

tRNA-guanine transglycosylase from Escherichia coli: recognition of dimeric, unmodified tRNA(Tyr).

In order to probe the interaction between tRNA and the tRNA hypermodifying enzyme, tRNA-guanine transglycosylase (TGT) from Escherichia coli, we have undertaken the generation of E coli tRNA(Tyr) and analogues. During efforts to adapt currently available in vitro transcription techniques we encountered difficulties attributable to dimerization of the tRNA products. E coli tRNA(Tyr) has previously been characterized for its ability to form a dimer in solutions of suitable salt concentrations at appropriate temperatures (Yang SK, Söll DG, Crothers DM (1972) Biochemistry 11, 2311-2320; Rordorff BF, Kearns DR (1976) Biochemistry 15, 3320-3330). We have applied similar techniques to our unmodified analogue of E coli tRNA(Tyr) and produced both monomeric and dimeric forms of E coli tRNA(Tyr). In this report we find that the dimer does serve as a substrate for modification by TGT. While both the conformers are equal in terms of Vmax (within experimental error) a 2.5-fold increase in KM occurs when going from monomer to dimer. This suggests that TGT preferentially binds the monomer but once either conformer is bound will catalyze the modification reaction equally well. We have also compared the results for the two conformers to our previous data of an RNA minihelix corresponding to the anticodon arm of E coli tRNA(Tyr). Here we find that our earlier conclusion, that the recognition elements for TGT are localized within the anticodon arm of cognate tRNAs, is supported.

tRNA-guanine transglycosylase from Escherichia coli: gross tRNA structural requirements for recognition.

tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the post-transcriptional modification of specific tRNAs (those for Asn, Asp, His, and Tyr) with the hypermodified base, queuine. In Escherichia coli this enzyme catalyzes the exchange of guanine-34 in the anticodon with preQ1, which is subsequently further modified to queuine. There is evidence that such hypermodified tRNA molecules may play a role in the control of cell proliferation and differentiation. In order to perform detailed, in vitro mechanistic studies and to probe the tRNA-enzyme interaction, we have generated unmodified E. coli tRNA(Tyr) and truncated analogues using an in vitro RNA synthesis system suggested by Milligan and Uhlenbeck [Milligan, J. F., & Uhlenbeck, O. C. (1989) Methods Enzymol. 180, 51-62]. From this system we have generated three tRNA analogues totally devoid of any post-transcriptional modifications. In order to compare the unmodified tRNA with the true physiological substrate for TGT, that is, tRNA that contains all modified bases except queuine, we have isolated E. coli tRNA(Tyr) from an overexpressing clone in a TGT-deficient strain of E. coli. We report here that unmodified, full-length tRNA(Tyr) serves as a substrate for TGT with kinetic parameters that are, within experimental error, the same as those for in vivo isolated tRNA(Tyr). This indicates that other post-transcriptional modifications have negligible effects upon TGT recognition of tRNA. A 17-base oligoribonucleotide, corresponding to the anticodon loop and stem, is also a substrate for TGT with only a 20-fold loss in Vmax/KM, versus the full-length tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)